The long-term objective of this research is to understand how an environmental bacterium gains the ability to transition from soil organism to life within mammalian cells. Listeria monocytogenes (Lm) is a facultative intra- cytosolic bacterium that is an increasingly important agent of serious food-borne infections and has been responsible for some of the largest food recalls in US history. The focus of this proposal is on an essential virulence regulatory protein known as PrfA that coordinates the complex and multifaceted Lm transition from life in the outside environment to life within an infected host. Critical to PrfA function is the shift from the low PrfA activity state found in bacteria replicatig outside of host cells to a high activity state following bacterial entry into the host cell. Cytosoic activation of PrfA has profound effects not only on the expression and secretion of Lm virulence gene products, but also on the metabolic state of Lm and on the ability of the bacterium to respond to conditions of stress. The central hypothesis of this proposal is that the regulation of PrfA activation is critical for Lm to successfully balance bacterial survival in the outside environment with life within mammalian cells. A combination of genetic, biochemical, and in vivo approaches will be used to elucidate the mechanisms that regulate PrfA activation status as well as to functionally decipher how PrfA activation coordinates different aspects of Lm stress resistance, bacterial cell signaling, and bacterial physiology to enhance bacterial growth and viability within the cytosol. Aim 1 will elucidate the mechanistic links between PrfA activation an different facets of Lm stress susceptibility and resistance. Experiments will clarify how PrfA activation selectively influences stress resistance to enhance bacterial survival within host cells and will identify targets of free radical damage that occur as a result of host inflammatory responses. The focus of Aim 2 is on mechanisms that promote bacterial viability in the cytosol under conditions of PrfA activation. Experiments will functionally define the contributions of two secretion chaperones and a pheromone-encoding lipoprotein known as Lmo2637 to pathogenesis; the secretion of these gene products is induced by PrfA activation and all have apparent roles in maintaining Lm viability within the cytosol. Aim 3 will clarify the molecular pathways leading to PrfA activation within the host and will seek to identify the cofactor that triggers PrfA activation. The ultimate goal of this proposal will be to elucidate the molecular pathways that promote Lm survival within host cells and the transformation of a soil dweller into a cell invader.